Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches

Translating Polyproline Translation of messenger RNA into protein is carried out by the ribosome, together with a variety of accessory factors, which offer the potential for regulation of this critical step in gene expression (see the Perspective by Buskirk and Green). Ude et al. (p. 82, published online 13 December), using bacterial genetics and an in vitro reconstituted translation system, and Doerfel et al. (p. 85, published online 13 December), using a model assay for peptide bond formation, find that the universally conserved bacterial elongation factor P (EF-P) (which is orthologous to the archaeal and eukaryotic initiation factor 5A) is required for the efficient translation of polyproline-containing polypeptides. Such short polyproline stretches (with runs of two, three, or more proline residues) would otherwise cause ribosomal stalling. A universally conserved translation factor facilitates synthesis of peptides that would otherwise cause ribosome stalling. [Also see Perspective by Buskirk and Green] Translation elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an elongation factor that enhances translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.

[1]  K. Jung,et al.  A comprehensive toolbox for the rapid construction of lacZ fusion reporters. , 2012, Journal of microbiological methods.

[2]  Daniel N. Wilson,et al.  Lys34 of translation elongation factor EF-P is hydroxylated by YfcM. , 2012, Nature chemical biology.

[3]  H. Aoki,et al.  Post-translational Modification by β-Lysylation Is Required for Activity of Escherichia coli Elongation Factor P (EF-P)* , 2011, The Journal of Biological Chemistry.

[4]  William Wiley Navarre,et al.  Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity , 2011, Journal of bacteriology.

[5]  M. Ibba,et al.  The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-β-lysine , 2011, Nature chemical biology.

[6]  V. Sharma,et al.  A mutation in the poxA gene of Salmonella enterica serovar Typhimurium alters protein production, elevates susceptibility to environmental challenges, and decreases swine colonization. , 2011, Foodborne pathogens and disease.

[7]  K. Jung,et al.  Identification of ArgP and Lrp as Transcriptional Regulators of lysP, the Gene Encoding the Specific Lysine Permease of Escherichia coli , 2011, Journal of bacteriology.

[8]  Ryohei Ishii,et al.  A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P , 2010, Nature Structural &Molecular Biology.

[9]  Runjun D. Kumar,et al.  PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica. , 2010, Molecular cell.

[10]  Daniel N. Wilson,et al.  Interplay between the ribosomal tunnel, nascent chain, and macrolides influences drug inhibition. , 2010, Chemistry & biology.

[11]  V. de Crécy-Lagard,et al.  Predicting the pathway involved in post-translational modification of Elongation factor P in a subset of bacterial species , 2010, Biology Direct.

[12]  Kirsten Jung,et al.  Induction kinetics of a conditional pH stress response system in Escherichia coli. , 2009, Journal of molecular biology.

[13]  T. Steitz,et al.  Formation of the First Peptide Bond: The Structure of EF-P Bound to the 70S Ribosome , 2009, Science.

[14]  Anthony C. Forster,et al.  Slow peptide bond formation by proline and other N-alkylamino acids in translation , 2009, Proceedings of the National Academy of Sciences.

[15]  M. Rodnina,et al.  Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates* , 2008, Journal of Biological Chemistry.

[16]  R. Heermann,et al.  Microbial Cell Factories Simple Generation of Site-directed Point Mutations in the Escherichia Coli Chromosome Using Red ® /et ® Recombination , 2022 .

[17]  Koreaki Ito,et al.  Peptidyl-prolyl-tRNA at the ribosomal P-site reacts poorly with puromycin. , 2008, Biochemical and biophysical research communications.

[18]  Kirsten Jung,et al.  The membrane‐integrated transcriptional activator CadC of Escherichia coli senses lysine indirectly via the interaction with the lysine permease LysP , 2008, Molecular microbiology.

[19]  D. Metzgar,et al.  Development of a novel continuous culture device for experimental evolution of bacterial populations , 2007, Applied Microbiology and Biotechnology.

[20]  M. Rasmussen,et al.  Identification of Salmonella enterica Serovar Typhimurium Genes Important for Survival in the Swine Gastric Environment , 2006, Applied and Environmental Microbiology.

[21]  J. Cronan A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control. , 2006, Plasmid.

[22]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[23]  Kirsten Jung,et al.  CadC-Mediated Activation of the cadBA Promoter in Escherichia coli , 2006, Journal of Molecular Microbiology and Biotechnology.

[24]  D. Newman,et al.  Anaerobic regulation by an atypical Arc system in Shewanella oneidensis , 2005, Molecular microbiology.

[25]  C. Beuzón,et al.  The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system. , 2003, Microbiology.

[26]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[27]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[28]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[29]  Trevor C. Charles,et al.  The chvH Locus ofAgrobacterium Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis , 2001, Journal of bacteriology.

[30]  Robert L. Lucas,et al.  Multiple Factors Independently RegulatehilA and Invasion Gene Expression in Salmonella enterica Serovar Typhimurium , 2000, Journal of bacteriology.

[31]  H. Aoki,et al.  Peptide Bond Synthesis: Function of the efp Gene Product , 2000, Biological chemistry.

[32]  R. Curtiss,et al.  Molecular and Functional Characterization ofSalmonella enterica Serovar Typhimurium poxAGene: Effect on Attenuation of Virulence and Protection , 1998, Infection and Immunity.

[33]  D. Lane,et al.  Expression of the second lysine decarboxylase gene of Escherichia coli. , 1998, Microbiology.

[34]  N. Kyrpides,et al.  Universally conserved translation initiation factors. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  N. W. Davis,et al.  The complete genome sequence of Escherichia coli K-12. , 1997, Science.

[36]  K. Jung,et al.  Purification, Reconstitution, and Characterization of KdpD, the Turgor Sensor of Escherichia coli * , 1997, The Journal of Biological Chemistry.

[37]  M. Neely,et al.  Kinetics of expression of the Escherichia coli cad operon as a function of pH and lysine , 1996, Journal of bacteriology.

[38]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[39]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[40]  C. Locht,et al.  Isolation and molecular characterization of a novel broad‐host‐range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram‐positive organisms , 1992, Molecular microbiology.

[41]  G. Bennett,et al.  Regulation of the Escherichia coli cad operon: location of a site required for acid induction , 1992, Journal of bacteriology.

[42]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[43]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[44]  H. Aiba,et al.  Evidence for two functional gal promoters in intact Escherichia coli cells. , 1981, The Journal of biological chemistry.

[45]  M. Ganoza,et al.  Peptide bond formation stimulated by protein synthesis factor EF-P depends on the aminoacyl moiety of the acceptor. , 1979, European journal of biochemistry.

[46]  G. Peterson,et al.  A simplification of the protein assay method of Lowry et al. which is more generally applicable. , 1977, Analytical biochemistry.

[47]  L. Enquist,et al.  EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: the lambdagtWES system. , 1977, Science.

[48]  M. Ganoza,et al.  Identification of a soluble protein that stimulates peptide bond synthesis. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[49]  W. Epstein,et al.  Potassium Transport Loci in Escherichia coli K-12 , 1971, Journal of bacteriology.

[50]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[51]  G. Bertani,et al.  STUDIES ON LYSOGENESIS I , 1951, Journal of bacteriology.

[52]  Jeffrey H. Miller A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Rela , 1992 .

[53]  N. Kleckner,et al.  Uses of transposons with emphasis on Tn10. , 1991, Methods in enzymology.

[54]  H. Rezvan,et al.  Conformational transitions of thromboplastin apoprotein from pig brain , 1979, FEBS letters.

[55]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .